From molecular hoovers and frameworks: A love story

Self assembly is a fascinating process and most importantly: it just works. Phospholipids assemble into a bilayer membrane in water, protecting their hydrophobic chains from the hydrophilic environment. Thus, combining membrane building blocks and membrane proteins in an aqueous environment should lead spontaneously to well-formed vesicles which carry the membrane protein of choice, resulting in a functional proteovesicle. Just like that! Love at first sight!

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Unfortunately, this love story does not end here but merely
starts.

Creating our molecular hoover, which is here a functionalized
vesicle which takes up a specific compound out of its environment
upon a trigger, requires the reconstitution of membrane proteins
which provide this functionality. But reconstitution of membrane proteins
is hard and can be, quoting one of our reviewers,

"considered
as an art rather than science by many working in this field".

When I joined the project and our collaborator Prof. Dimitrios
Fotidias' group, work was focused on the insertion of proteorhodopsin
(PR) into liposomes. Even though the reconstitution seemed
successful, we could not measure any proton pumping activity. As we
were working with the wild type protein, the protein’s orientation
was probably the issue (and later solved via a chemical switch [1]).
I continued my work focusing on polymer membranes and followed the
common protocols for membrane protein reconstitution into polymer
membranes. All trials were unsuccessful. Adding the membrane protein
to a polymer-ethanol mixture, removing the solvent and rehydrate the
polymer-protein film can work for ultra-stable membrane proteins like
OmpF [2].
However, not for more delicate proteins like PR. As literature about
the reconstitution of PR in polymer membranes was rare at that point,
we briefly (around 1.5 PhD years) worked on the adaption of
reconstitution protocols from lipid membranes to polymer membranes.
But just trying different detergent and polymer combinations as well
as more exotic methods like GRecon [3]
did not lead to reliable and reproducible results.

This outlook of one of my first presentations in this project showed some promising results using the GRecon method. Unfortunately, the experiments never succeeded.

When one wants to engineer a functional system, one combines
several known parts in a certain manner, based on the desired goal
and the knowledge about each part. When you want to build car, you
assemble the engine, the chassis, the tires, etc. Each part is known
and you can predict its behavior and the interplay with the other
parts, for example via simulations. In biology, more often than not,
we are not able to do that because we lack the knowledge and the
theoretical framework. There is no general equation which describes
the reconstitution process of proteorhodopsin.

Within our publication we did not solve that but with the help of
design of experiments (DoE) we derived a working model for our
specific problem: How to carry out a functional reconstitution of PR
into a polymer membrane. DoE helps a lot if you need to investigate a
process which depends on several parameters and you are not sure
which parameter has an effect and how strong that one is (like making
good coffee, unfortunately German only).

Together with the creation of a PR
fusion protein, which has green fluorescent protein (GFP) attached,
by my colleagues Johannes Thoma and Noah Ritzmann from Prof. Daniel
Müller’s group [4],
we were able to tackle the project systematically. PR-GFP solved two
problems: 1st the orientation of the membrane protein is fixed and 2nd
we could detect the membrane protein after reconstitution reliably.
DoE provided the necessary framework and for the first time in our
lab we could pinpoint important factors, estimate their influence and
predict the assembly of our proteovesicles under varying conditions.
Because we used both lipid and polymer membranes, we could compare the
two systems and identify the differences, like the necessity of basic
pH values for proteopolymersome formation. Furthermore, we found that
our proteopolymersomes carry less proton pumps than the
proteoliposomes but generate a similar pH gradient, making the more
efficient. Even though this phenomenon needs more investigation, it is likely to be attributed to the higher thickness of polymer
membranes which makes them less permeable to the back-diffusion of
pumped protons. For me personally, the computational assistance
deployed in the later stages of the project was equally important.
Using DoE is relatively easy nowadays, thanks to specialized programs
which I used in the beginning. However, the use of the statistical
programming language R to write my own scripts for the data
assessment was a major milestone as it forced me to get directly in touch with my data.
It was also the first programming language I learned and I would like
to encourage people to give programming languages like R or Python a
serious try as they might change the way one thinks about data.

A schematic presentation of the vision behind that work. Creating molecular factories, where several functional proteovesicles work together.

Beside the obtained results in this publication, this work could
lay the foundation of a data library for membrane protein
reconstitution in our labs. If the design and its experiments would be repeated
for more membrane proteins, detergents and membrane types, we could
add factors like the detergent type, structural features of the
membrane proteins (e.g. alpha-helical, beta-sheets) to the model as
categorical factors. With the additional data points it should be
possible to determine conditions which, for example, are more
suitable for one class or the other and thus ease future
reconstitutions. This information will be crucial if we want to
design and build up complex systems like molecular factories.

For me, the love story ends here. My relationship with PR and
vesicles has been a difficult one and we had our ups and downs. But
the result that DoE can provide insight into the underlying
mechanisms of membrane protein reconstitution is reassuring and might
change the balance more towards science than art.

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